Structural and Functional Insights into Foamy Viral Integrase (original) (raw)
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3 '-Processing and strand transfer catalysed by retroviral integrase in crystallo
EMBO Journal, 2012
Retroviral integrase (IN) is responsible for two consecutive reactions, which lead to insertion of a viral DNA copy into a host cell chromosome. Initially, the enzyme removes di-or trinucleotides from viral DNA ends to expose 3 0hydroxyls attached to the invariant CA dinucleotides (3 0 -processing reaction). Second, it inserts the processed 3 0 -viral DNA ends into host chromosomal DNA (strand transfer). Herein, we report a crystal structure of prototype foamy virus IN bound to viral DNA prior to 3 0 -processing. Furthermore, taking advantage of its dependence on divalent metal ion cofactors, we were able to freeze trap the viral enzyme in its ground states containing all the components necessary for 3 0 -processing or strand transfer. Our results shed light on the mechanics of retroviral DNA integration and explain why HIV IN strand transfer inhibitors are ineffective against the 3 0 -processing step of integration. The ground state structures moreover highlight a striking substrate mimicry utilized by the inhibitors in their binding to the IN active site and suggest ways to improve upon this clinically relevant class of small molecules.
Prototype Foamy Virus Integrase Displays Unique Biochemical Activities among Retroviral Integrases
Biomolecules, 2021
Integrases of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast with other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 s, PFV intasomes do not commit to target DNA during their reaction lifetime. Together, these data high...
Proceedings of the National Academy of Sciences, 2000
Insolubility of full-length HIV-1 integrase (IN) limited previous structure analyses to individual domains. By introducing five point mutations, we engineered a more soluble IN that allowed us to generate multidomain HIV-1 IN crystals. The first multidomain HIV-1 IN structure is reported. It incorporates the catalytic core and C-terminal domains (residues 52-288). The structure resolved to 2.8 Å is a Y-shaped dimer. Within the dimer, the catalytic core domains form the only dimer interface, and the C-terminal domains are located 55 Å apart. A 26-aa ␣-helix, ␣6, links the C-terminal domain to the catalytic core. A kink in one of the two ␣6 helices occurs near a known proteolytic site, suggesting that it may act as a flexible elbow to reorient the domains during the integration process. Two proteins that bind DNA in a sequence-independent manner are structurally homologous to the HIV-1 IN C-terminal domain, suggesting a similar protein-DNA interaction in which the IN Cterminal domain may serve to bind, bend, and orient viral DNA during integration. A strip of positively charged amino acids contributed by both monomers emerges from each active site of the dimer, suggesting a minimally dimeric platform for binding each viral DNA end. The crystal structure of the isolated catalytic core domain (residues 52-210), independently determined at 1.6-Å resolution, is identical to the core domain within the two-domain 52-288 structure.
The mechanism of retroviral integration from X-ray structures of its key intermediates
Nature, 2010
To establish successful infection, a retrovirus must insert a DNA replica of its genome into host cell chromosomal DNA 1 , 2 . This process is carried out by the intasome, a nucleoprotein complex comprised of a tetramer of integrase (IN) assembled on the viral DNA ends 3 , 4 . The intasome engages chromosomal DNA within a target capture complex to carry out strand transfer, irreversibly joining the viral and cellular DNA molecules. Although several intasome/ transpososome structures from the DDE(D) recombinase superfamily were reported 4 -6 , the mechanics of target DNA capture and strand transfer by these enzymes have not been established. Herein, we report crystal structures of the intasome from prototype foamy virus in complex with target DNA, elucidating the pre-integration target DNA capture and post-catalytic strand transfer intermediates of the retroviral integration process. The cleft between IN dimers within the intasome accommodates chromosomal DNA in a severely bent conformation, allowing widely spaced IN active sites to access the scissile phosphodiester bonds. Our results elucidate the structural basis for retroviral DNA integration and moreover provide a framework for the design of INs with altered target sequences.
Virology, 1995
The retroviral integrase (IN) is a virus-encoded enzyme that is essential for insertion of viral DNA into the host chromosome. In order to map and define the properties of a minimal functional domain for this unique vira] enzyme, a series of N-and C-terminal deletions of both Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV) INs were constructed. The RSV IN deletion mutants were first tested for their ability to remove two nucleotides from the end of a substrate representing the terminus of viral DNA in order to assess the contribution of N and C regions towards this reaction, referred to as processing. The results suggest that C-terminal amino acids of the intact RSV protein are required to maintain specificity of the processing reaction. Though deficient for processing, the RSV deletion mutants exhibited a secondary endonucleolytic activity that was indistinguishable from that of wild-type IN, demonstrating that all retained some enzymatic activity. RSV, and a larger set of HIV-1, IN deletion mutants were then tested for their ability to perform an intramolecular, concerted cleavage-ligation reaction using an oligodeoxynucleotide substrate that mimics the intermediate viral-host DNA junction found prior to the final step of covalent closure. The composite results from such analyses define a minimal functional central region of-140 amino acids for each enzyme that includes the highly conserved D,D(35)E domain. Results with HIV-1 and HIV-2 IN also indicate that the efficiency of concerted cleavage-ligation depends upon the presence of CA/GT base pairs within the viral component of the DNA substrate at the reaction site. Even the isolated central region of HIV-1 IN exhibited this sequence requirement for optimal activity. We conclude that this evolutionarily conserved central region of IN not only encodes residues that are required for the catalytic activity of the enzyme but also harbors some or all of the determinants responsible for recognition of the CA/GT dinucleotides that are present at the ends of all retroviral DNAs.
Major and Minor Groove Contacts in Retroviral Integrase−LTR Interactions
Biochemistry, 1999
The 3′-processing activities of HIV-1, HTLV-2, and M-MuLV integrases (INs) with their corresponding U5 end of the viral DNA molecule were examined to define functional group determinants of U5 terminus recognition and catalysis. Nucleotide analogues were incorporated into the U5 terminus to produce conservative modifications in the surface of the major and/or minor grooves to map the hydrogenbonding contacts required for LTR-IN interaction. Specifically, the phylogenetically conserved CA (positions 4 and 3, respectively) and the 5′-proximal nucleotide (position 5) were replaced with base analogues in plus and/or minus strands. For each integrase, similar major and minor groove contacts were identified in the guanine and adenine of the conserved CA/GT. Overall, perturbances in the minor groove resulted in a greater decrease in 3′-processing activity than the major groove substitutions. Additionally for HIV-1 and HTLV-2 INs, we observed an increase in the 3′-processing activity with an O 4 -MeThy substitution at position 3 of the minus strand. O 4 -MeThy may act to destabilize Watson-Crick base pairing and in doing so provide these INs with a more favorable interaction with the adjacent scissile bond. At position 5, a substantial divergence among the three INs was noted in the functional groups required for 3′-processing activity, thereby supporting the role of this position in providing some level of substrate specificity.
PLoS ONE, 2011
Background: We applied crosslinking techniques as a first step in preparation of stable avian sarcoma virus (ASV) integrase (IN)-DNA complexes for crystallographic investigations. These results were then compared with the crystal structures of the prototype foamy virus (PFV) intasome and with published data for other retroviral IN proteins. Methodology/Results: Photoaffinity crosslinking and site-directed chemical crosslinking were used to localize the sites of contacts with DNA substrates on the surface of ASV IN. Sulfhydryl groups of cysteines engineered into ASV IN and aminomodified nucleotides in DNA substrates were used for attachment of photocrosslinkers. Analysis of photocrosslinking data revealed several specific DNA-protein contacts. To confirm contact sites, thiol-modified nucleotides were introduced into oligo-DNA substrates at suggested points of contact and chemically crosslinked to the cysteines via formation of disulfide bridges. Cysteines incorporated in positions 124 and 146 in the ASV IN core domain were shown to interact directly with host and viral portions of the Y-mer DNA substrate, respectively. Crosslinking of an R244C ASV IN derivative identified contacts at positions 11 and 12 on both strands of viral DNA. The most efficient disulfide crosslinking was observed for complexes of the ASV IN E157C and D64C derivatives with linear viral DNA substrate carrying a thiol-modified scissile phosphate.